This application is for a Transition Career Development Award for Dr. Carlos de los Santos, who is a junior faculty member in the Department of Pharmacological Sciences at SUNY Stoney Brook. Dr. de los Santos is a structural biologist working in the field of DNA damage and is in the last year of Career Development Award (K01) from the NCI. The applicant's long-term career goal is to understand processes involved in the recognition and repair of DNA lesions at the molecular level, and to correlate these processes with chemical and environmental mutagenesis and carcinogenesis. The present application involves the study of Multiply Damaged Sites (MDS) in DNA. A unique property of ionizing radiation and some radiomimetic chemotherapeutic drugs is the production of clustered DNA damage, this is two or more DNA lesions (oxidized bases, modified sugars, single (SSB) and double strand breaks (DSB)) located within a single turn of the DNA helix. It has been known for some time that the number of DSB correlates directly with the kill effects of ionizing radiation. Recently, it has been shown that, in addition to DSB, MDS composed of base and/or sugar damages are readily produced in the cell after low doses of ionizing radiation, and that they make up to 80% of the total clustered damage. Attempts to repair MDS can produce different outcomes, depending on the type of lesions, their separation, and relative orientation. Glycosylase activity studies using purified enzymes or nuclear cell extracts showed that some MDS can be cleaved readily generating toxic DSB, while others are incised very poorly, persisting in the cell for longer periods of time. Furthermore, MDS composed by identical lesions can be processed differently depending on damage separation and/or relative orientation. At the present time, the structural basis that explains this property is almost non-existent. We are currently using high-resolution NMR spectroscopy and restrained molecular dynamics simulations to determine three-dimensional structures of DNA duplexes containing representative MDS. In order to correlate the structures with biological function, we propose to determine recognition and repair properties of model clustered lesions. We will investigate recognition and processing of MDS by purified BER proteins and in the presence of nuclear cell extracts. We will examine base excision repair of MDS using eukaryotic nuclear cell extracts to establish the extent and hierarchy of repair. We will isolate nuclear cell proteins that bind these lesions and establish their identity by mass spectroscopy methods. Completion of this proposal would establish relationships between the solution structure of clustered bistrand lesions and some of their biological properties. Additionally, it would afford additional time to the applicant for the establishment of an independent research program in his laboratory.